Genetic analysis of the succinate utilization genes in Pseudomonas fluorescens SBW25 : a thesis presented in fulfillment of the requirements for the degree of Master of Philosophy (Science) in Microbiological Genetics at Massey University, Auckland, New Zealand

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Succinate, an intermediate of the tricarboxylic acid (TCA) cycle, is one of the most preferred nutritional substrates for bacteria, particularly those capable of colonizing eukaryotic hosts such as plants, animals (including humans). The genetic mechanisms of succinate utilization have been well studied in E. coli and other model microorganisms such as rhizobia, a group of gram-negative bacteria that form nitrogen-fixing nodules on leguminous plants. Uptake of succinate is mediated by the DctA transporter, whose expression is regulated by the twocomponent signal transduction system DctB / DctD in a succinate dependent manner. In the presence of succinate, the DctB sensor kinase binds to succinate, causing phosphorylation of the response regulator DctD that in turn activate transcription of dctA with the help of the alternative sigma factor s54. Our work on the genetics of succinate utilization has focused on Pseudomonas fluorescens SBW25. P. fluorescens SBW25 is a plant growth-promoting bacterium that was originally isolated from the phyllosphere of sugar beet plants. When colonizing on the surfaces of sugar beet, P. fluorescens SBW25 activates the expression of a suite of genes involved in nutrient acquisition, including pflu4717 with a predicted role in succinate uptake. The deduced amino acid sequence of pflu4717 shows 70% sequence identity with dctA from E. coli, and 63% with dctA of Sinorhizobium meliloti 1021. To confirm the predicted role of pflu4717 in succinate uptake, a pflu4717 deletion mutant was constructed and the resultant mutant strain was unable to grow on succinate as the sole source of carbon and energy (Suc-). The inability of the pflu4717 mutant to grow on succinate can be restored by the introduction of a cloned copy of pflu4717. Furthermore, expression of pflu4717 was induced by the presence of succinate as measured by using an integrated lacZ reporter gene. Together, the data consistently indicate that pflu4717 encodes DctA for succinate uptake, and it is thus named dctA. Next, we sought to identify the transcriptional regulators of dctA in P. fluorescens SBW25. In silico analysis was performed using the DctBD sequences of Sinorhizobium meliloti 1021. The analysis identified three pairs of two-component regulatory systems: Pflu0287/Pflu0286, Pflu4953/Pflu4954 and Pflu1135/Pflu1134. However, deletion analysis for each of the three response regulators (Pflu0286, Pflu4954 and Pflu1134) showed that only the deletion mutant of pflu0286 lost the ability to grow on succinate; and moreover, expression of dctA was not responsive to succinate in the growth medium. The data thus showed that pflu0287 / pflu0286 encode the DctB / DctD required for the succinate-induced expression of dctA in P. fluorescens SBW25. Whilst the dctA deletion mutant (SBW25?dctA) cannot grow on minimal medium supplemented with succinate as the sole carbon source, interestingly, a spontaneous Suc+ mutant arose at high frequency (~ 10-4). To identify the suppressor mutations, two such spontaneous Suc+ mutants were subject to genome re-sequencing, which led to the identification of two separate mutations in a putative sensor kinase Pflu4953. Pflu4953 forms a two-component regulatory system with Pflu4954, but as has been shown above is not involved in the utilization of succinate. Next, a logic series of experiments were performed using a combination of sitedirected mutagenesis analysis and ß-galactosidase assays. The results led to the conclusion that: (1) Pflu4953 and Pflu4954 (designated DctX and DctY, respectively here) regulate the expression of a putative transporter Pflu4955 (designated DctT); (2) DctT is responsible for the uptake of alpha-ketoglutarate (another intermediate of the TCA cycle), but it is also capable of transporting succinate; (3) however, the DctXY-mediated expression of dctT is induced by alphaketoglutarate, and not by succinate; (4) mutation of DctX caused constitutive expression of DctT, which enables the ?dctA mutant to grow on succinate (Suc+). Taken together, the data show that P. fluorescens SBW25 possesses two transporter systems for the uptake of succinate (i.e., DctA and DctT), which are regulated by the DctBD and DctXY two-component systems, respectively. However, the primary role of DctT is for the uptake of alpha-ketoglutarate and not succinate, as expression of DctT is only induced by alphaketoglutarate. This finding indicates that substrate specificity of an uptake system is determined by not only the transporter protein but also its regulator(s). Given that succinate is significant nutrient available on the plant surfaces, the encoded multiple systems for succinate uptake likely contribute to the success of P. fluorescens SBW25 in the plant environment.
Pseudomonas fluorescens, Genetics, Nutrition